This application is aimed at validating a regulatory function for an array of cis-acting elements in the 5'untranslated region of the gamma globin gene and cognate transcription factors including Stat3beta in gamma globin silencing. The collection of cis elements includes three Stat3 DNA elements at -14, +9 and +26, one GATA-1 element at +26 previously assigned a negative regulatory function, and a putative HoxB2 binding AT-rich element at +26. We have recently demonstrated by site directed mutagenesis and co-transfecfion analysis that the +9 cis-element is required for Stat3beta-mediated repression of the gamma globin promoter. Furthermore, subsequent experiments presented as preliminary data in this application show recombinant Stat3 binding to the +9 and +26 cis elements in vitro. We next explored the possibility that Stat3beta inhibits gamma globin promoter via protein-protein interactions with other transcription factors such as HoxB2, a known inhibitor of gamma promoter activity. We found potential interactions between Stat3 with HoxB2, GATA-1 and CBP. This suggests that inhibition of the gamma globin by Stat3beta may involve synergy with HoxB2 and functional antagonism with GATA-1. Based on these preliminary observations we hypothesize that "a Stat3-containing multi-protein complex interacts through a putative gamma-globin silencing domain to repress the gamma gene". This hypothesis will be tested in four specific aims, 1) Determine the functional significance of cis elements within a putative gamma-globin silencing domain by mutation analysis in chromosomally integrated g promoters. 2) Demonstrate in vivo Stat3 binding to the putative gamma-globin silencing domain and identify components of the Stat3 repressor complex by chromatin immunoprecipitation and mass spectrometry. 3) Develop methods to inhibit Stat3-mediated gamma globin repression as a therapeutic strategy for inducing HbF production by two approaches. The first approach will test the ability of Stat3cz to functionally inhibit Stat3 gamma. Secondly we will test the ability of novel anti-Stat3 binding peptides to de-repress the y gene promoter. 4) Finally we will test the efficacy of combined treatment with activated Stat3 blocking peptides and novel short chain fatty acids to augment g gene expression. This project thus offers a systematic analysis of gamma globin silencing that offers a unique opportunity to develop a novel gene-based therapy to increase HbF expression in Sickle cell disease and Cooley's anemia.